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US 2026/0132172 A1Published application
In vitro biosynthesis of diverse pyridine-based macrocyclic peptides
AssigneeThe Brd. of Trustees of the Univ. of Illinois
InventorsDouglas Alan Mitchell, Dinh Thanh Nguyen, Wilfred A. Van Der Donk
Life of this patent
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Abstract
Provided herein are compositions and methods for producing pyridine-based macrocyclic peptides.
Claims21
A substrate for enzyme synthesis of pyridine-based macrocyclic peptides comprising a leader region and a core region, wherein the leader region comprises:
- (SEQ ID NO:
- 1)
- X1LDX2X3X4X5X6LX7X8X9X10X11LX12X13X14X15X16X17GLGNTEVGA,
- wherein
- X1 is D, S or A;
- X2 is I or V;
- X3 is V, T, M, or A;
- X4 is D, N, or T;
- X5 is L or V;
- X6 is D or E;
- X7 is A or P;
- X8 is V, I, or G;
- X9 is D, E, or S;
- X10 is E or D;
- X11 is E, L, V, or absent;
- X12 is A or V;
- X13 is A, E, or K;
- X14 is L, V, or A;
- X15 is S, L, or V;
- X16 is V, I, G, T, or A;
- X17 is G or M;
- and
- wherein the core region comprises:
- (SEQ ID NO:
- 10)
- SGX1SX4X2X3,
- wherein X1 is three to twenty amino acids, and wherein X2 is V or L, wherein X3 is I or V, wherein X4 is Y, W, F, or H, and wherein the leader and core can be separate polypeptides used in combination, a single fusion protein, or covalently linked polypeptides.
24 more elements
A fusion protein comprising:
- (a) Micromonospora dehydratase (MroB or MroC or both MroB and MroC) and an affinity tag;
- or
- (b) Micromonospora macrocyclase (MroD) and an affinity tag.
A substrate for enzyme synthesis of pyridine-based macrocyclic peptides comprising a leader region of:
- MDNVVTEAAEFADLDIDDFDLAVDEELAALSVGGLGNTEVGA (SEQ ID NO:36).
- and core sequence can be SCNCFCYICCSX1LI (SEQ ID NO:37), wherein X1 is Y, W, F, or H, or SCX2CX2CX2ICCSX1LI (SEQ ID NO:43), wherein X1 is Y, W, F, or H, and wherein X2 is any amino acid;
- or
- (SEQ ID NO:
- 36)
- MDNVVTEAAEFADLDIDDFDLAVDEELAALSVGGLGNTEVGA,
- and
- a core sequence of SX1N X1F X1YI X1 X1SX2LI, (SEQ ID NO:38) wherein X2 is Y, W, F, or H, wherein each of X1 are thiazole, thiazoline, oxazole, oxazoline, methyloxazole, methyloxazoline, or combinations thereof or a core sequence of SX1X3 X1X3 X1X31 X1 X1SX2LI, (SEQ ID NO:
- 44) wherein X2 is Y, W, F, or H, wherein each of X1 are thiazole, thiazoline, oxazole, oxazoline, methyloxozole methyloxazole, methyloxazoline, or combinations thereof, and wherein X3 is any amino acid, and wherein the leader region and core can be separate polypeptides used in combination, a single fusion protein, or covalently linked polypeptides.
6 more elements
A method of making pyridine-based macrocyclic peptides comprising contacting a first substrate comprising:
- a leader region of
- (SEQ ID NO:
- 36)
- MDNVVTEAAEFADLDIDDFDLAVDEELAALSVGGLGNTEVGA,
- and
- a core sequence of SCNCFCYICCSX1LI (SEQ ID NO:37), wherein X1 is Y, W, F, or H, or SCX2CX2CX2ICCSX1LI (SEQ ID NO:43), wherein X1 is Y, W, F, or H, and wherein X2 is any amino acid,
- with thiazole synthetase, TbtE, TbtF, TbtG, or TbtD such that a second substrate is formed comprising:
- a leader region of
- (SEQ ID NO:
- 36)
- MDNVVTEAAEFADLDIDDFDLAVDEELAALSVGGLGNTEVGA,
- and a core sequence of SX1N X1F X1YI X1 X1SX2LI, (SEQ ID NO:38) wherein X2 is Y, W, F, or H, wherein each of X1 are thiazole, thiazoline, oxazole, oxazoline, methyloxazole, methyloxazoline, or combinations thereof, or a core sequence of SX1X3 X1X3 X1X31 X1 X1SX2LI, (SEQ ID NO:44) wherein X2 is Y, W, F, or H,
- wherein each of X1 are thiazole, thiazoline, oxazole, oxazoline, methyloxazole, methyloxazoline, or combinations thereof, and wherein X3 is any amino acid,
- wherein the leader region and core sequence can be separate polypeptides used in combination, a single fusion protein, or covalently linked polypeptides;
- and contacting the second substrate with MroB, MroC, and MroD such that pyridine-based macrocyclic peptides are made.
12 more elements
-41. (canceled)
Dependencies are inferred from the claim text.